1a.Objectives (from AD-416):
Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen-specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors.

1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus.
2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage.
3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus.

1b.Approach (from AD-416):
Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype-specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California.

3.Progress Report:
Research in this project was initiated in April 2012 to replace the previous project (5302-22000-009-00D) which terminated. FY12 milestones are, therefore, in progress. Success was achieved in Milestones 1 (pathogen target capture to membranes); Milestone 2 (cDNA and small RNA libraries for Tristeza-infected cross-protection tests); Milestone 5 and 6 (New tristeza isolates or strains were collected in 2012 characterizations and incorporation into cross-protection tests initiated. For Milestones not met, aphid and leafhopper vector colonies are being developed for vigor and abundance to start vector transmission tests in fall when greenhouse temperature is more favorable for pathogen multiplication (Milestone 3 &.4)and new pathogen collections can be made from field sources (Milestone 4).

4.Accomplishments
1.
Cross-protection of Citrus tristeza virus. The mechanism and duration of effective ross-protection (where a mild isolate of the virus can protect a plant against disease development caused by a severe isolate of the virus) of tristeza are unknown. ARS researchers at Parlier, California, in collaboration with scientists at Consiglio Nazionale Delle Ricerche, Bari, Italy, examined RNA interference (RNAi) as a natural plant defense system for cross-protection of a severe virus strain. Profiling of small RNAs from sour orange plants infected by different combinations of mild and severe tristeza strains revealed defined small RNAs were correlated with plants showing mild or no symptoms. Further profiling revealed 40 microRNA families with different levels of expression in plants and suggested degradation of tristeza strain. These data suggest RNAi was active in tristeza cross-protection and that small viral RNAs or plant microRNAs may be useful as biomarkers to identify cross-protective tristeza strains.

2.
Next generation sequencing of Citrus Tristeza Virus. Cross-protection of tristeza that occurred with a mixture of tristeza genotypes was different from cross-protection by exclusion of a strain within the same genotype. ARS researchers at Parlier, California, in collaboration with scientists at Consiglio Nazionale Delle Ricerche, Bari, Italy, achieved ~95% full-length genome sequencing of a mild cross-protective Citrus Tristeza Virus (CTV) strain (Dek-NS) using deep sequencing technologies. Complete sequence of mild cross-protective tristeza strains complements studies of RNAi and host microRNA-mediated regulation of gene expression in relation to disease symptomology. Indepth analysis of molecular interactions between citrus and tristeza strain(s) should lead to improved selection of cross-protective tristeza strains to control severe strains of CTV.